Transgenic plants with a modified activity of a plastidial ADP/ATP translocator

ABSTRACT

Transgenic plant cells and plants are described which, compared to wildtype cells and plants, exhibit an increased yield, in particular, an increased oil and/or starch content and which preferably synthesize a modified starch. The described plants exhibit an increase or a decrease of the plastidial ADP/ATP translocator activity.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/EP99/03292 which has an Internationalfiling date of May 12, 1999, which designated the United States ofAmerica.

The present invention relates to transgenic plant cells and plants withan increased plastidial ADP/ATP translocator activity. Such cells andplants exhibit an increased yield, preferably an increased oil and/orstarch content, and synthesize preferably a starch with increasedamylose content.

Further, the present invention relates to transgenic plant cells andplants with a decreased ADP/ATP translocator activity. Such cells andplants synthesize a starch with decreased amylose content.

In the field of agriculture and forestry there have been permanentendeavours to provide plants with an increased yield, in particular, inorder to ensure the food supply of the permanently growing worldpopulation and to guarantee the supply of regenerating raw materials.Traditionally, it has been tried to obtain high-yield plants bybreeding. This, however, is time-consuming and costly. Furthermore,corresponding breeding programs have to be carried out for each plantspecies of interest.

Progress has been made, partly, by genetic manipulation of plants, i.e.by purposeful introduction and expression of recombinant nucleic acidmolecules in plants. Approaches of that kind have the advantage thatthey are, in general, not limited to one plant species but can betransferred to other plant species, too.

In EP-A 0 511 979, for example, it was described that the expression ofa prokaryotic asparagin synthetase in plant cells leads to an increasedbiomass production, amongst others. WO 96/21737 describes, for example,the increase in yield of plants by expression of de- or unregulatedfructose-1,6-bisphosphatase due to increase in the rate ofphotosynthesis. Nevertheless, there is still a need for generallyapplicable methods for the improvement of the yield in plantsinteresting for agriculture or forestry.

Furthermore, with regard to the fact that substances contained in plantsplay a more and more important role as renewable sources of rawmaterial, one of the problems in biotechnological research is theadjustment of said vegetable raw materials to the requirements of theprocessing industry. In order to allow for application of regeneratingraw materials in as many fields as possible it is furthermore necessaryto achieve a wide range of substances. Moreover, it is necessary toincrease the yield of said vegetable content substances in order toincrease the efficiency of the production of renewable sources of rawmaterial from plants.

Apart from fats and proteins, oils and polysaccharides are the essentialregenerating vegetable raw materials. A central role with thepolysaccharides, apart from cellulose, plays starch which is one of themost important reserve substances in higher plants. Amongst those,potato and maize, in particular, are interesting plants since they areimportant cultivated plants for the production of starch.

The polysaccharide starch which is one of the most important reservesubstances in the vegetable world is, apart from its use in the foodindustry, widely used as regenerating raw material for the production ofindustrial products.

The starch industry has a great interest in plants with increased starchcontent which, as a rule, means an increased dry weight. An increaseddry weight increases the value of the plants processed in the starchindustry (maize, potato, tapioca, wheat, barley, rice etc.) due to theincreased yield of starch. In addition, plant cells or organs containinghigher amounts of starch offer advantages for the processing in the foodindustry since they absorb less fat or frying oil and, thus, lead to“healthier” products with reduced caloric content. Said property is ofgreat importance e.g. in the production of popcorn, corn flakes frommaize or chips, crisps or potato fritters from potatoes.

For the industry processing potato starch the dry weight (starchcontent) is a crucial size since it determines processing costs. Anincreased dry weight (starch content) means, that with the same yield,the water content of the potato tuber is reduced. The reduced watercontent leads to reduced transport costs and to a reduction of the exactcooking period necessary in cooking.

Therefore, it seems desirable to provide plant cells and plantsexhibiting an increased starch content as well as methods for theproduction of such plant cells and plants. Moreover, it seems desirableto provide starches whose amylose and amylopectin content meets therequirements of the processing industry. In this context, both starcheswith an increased amylose content and starches with a reduced amylosecontent are of interest since they are particularly suitable for specialuses each.

Thus, the problem underlying the present invention is to provide plantcells and plants which, in comparison with corresponding non-modifiedwild type plant cells and wild type plants, exhibit an increased yieldpreferably of oil and/or starch and/or synthesize a starch with amodified amylose content.

This problem is solved by the provision of the embodiments characterizedin the claims.

Thus, the present invention relates to transgenic plant cells which aregenetically modified, wherein the genetic modification is theintroduction of a foreign nucleic acid molecule whose presence orexpression leads to an increase in the plastidial ADP/ATP translocatoractivity in the transgenic cells in comparison with correspondinggenetically non-modified plant cells from wild type plants.

In this context, the genetic modification can be any geneticmodification leading to an increase in the plastidial ADP/ATPtranslocator activity. One possibility, for example, is the so-called“in situ-activation”, wherein the genetic modification is a change ofthe regulatory regions of endogenous ADP/ATP translocator genes, whichleads to an increased expression of said genes. This can be achieved,for example, by means of introduction of a very strong promoter in frontof the corresponding genes, e.g. by means of homologous recombination.

Further, there is the possibility to apply the method of the so-called“activation tagging” (cf. e.g. Walden et al., Plant J. (1991), 281-288;Walden et al., Plant Mol. Biol. 26 (1994), 1521-1528). Said method isbased on the activation of endogenous promoters by means of enhancerelements such as the enhancer of the ³⁵S RNA promoter of the cauliflowermosaic virus or the octopin synthase enhancer.

In a preferred embodiment the genetic modification comprises, however,the introduction of a foreign nucleic acid molecule encoding aplastidial ADP/ATP translocator into the genome of the plant cell.

The term “transgenic”, therefore, means that the plant cell of theinvention contains at least one foreign nucleic acid molecule encoding aplastidial ADP/ATP translocator stably intergrated in the genome,preferably a nucleic acid molecule.

The term “foreign nucleic acid molecule” preferably means a nucleic acidmolecule which encodes a protein with the biological activity of aplastidial ADP/ATP translocator and either does not occur naturally incorresponding plant cells or does not occur naturally in the precisespatial order in the plant cells or which is localized at a place in thegenome of the plant cell where it does not occur naturally. Preferably,the foreign nucleic acid molecule is a recombinant molecule whichconsists of various elements and whose combination or specific spatialarrangement does not occur naturally in plant cells. The transgenicplant cells of the invention contain at least one foreign nucleic acidmolecule encoding a protein with the biological activity of a plastidialADP/ATP translocator, wherein said nucleic acid molecule preferably isconnected with regulatory DNA elements ensuring the transcription inplant cells, in particular with a promoter.

In principle, the foreign nucleic acid molecule can be any nucleic acidmolecule encoding an ADP/ATP translocator which, after expression, islocalized in the inner membrane of plastids. In this context, aplastidial ADP/ATP translocator is a protein catalyzing the transport ofATP into the plastids and of ADP out of the plastids. Such nucleic acidmolecules are known, for example, from Arabidopsis thaliana (Kampfenkelet al., FEBS Lett. 374 (1995), 351-355; Genebank Acc. No. X94626 andAcc. No. Z49227) or from potato (Genebank Acc. No. Y10821). By means ofsaid known nucleic acid molecules the person skilled in the art canisolate corresponding sequences from other organisms, particularlyvegetable ones, according to standard methods, for example byheterologous screening. In particular, non-vegetable nucleic acidmolecules can be used, too, which encode an ADP/ATP translocator and areconnected with a targeting sequence ensuring the localisation in theinner plastid membrane. In this context, e.g. an ADP/ATP translocator isknown from Rickettsia prowazekii (Williamson et al., Gene 80 (1989),269-278) and from Chlamydia trachomatis.

In a preferred embodiment the foreign nucleic acid molecule encodes aplastidial ADP/ATP translocator form Arabidopsis thaliana, in particularthe protein AATP1 described in Kampfenkel et al. (1995, loc. cit.).

The cells of the invention can be distinguished from naturally occurringplant cells, amongst others, in that they contain a foreign nucleic acidmolecule which does not occur naturally in said cells or in that saidmolecule is integrated at a place in the genome of the cell where itdoes not normally occur, i.e. in another genomic environment. Further,said transgenic plant cells of the invention can be differentiated fromnaturally occurring plant cells in that they contain at least one copyof the foreign nucleic acid molecule stably integrated in their genome,optionally in addition to the copies of said molecule naturallyoccurring in the cells. If the nucleic acid molecule(s) introduced intothe cells is/ (are) an additional copy(ies) of molecules naturallyoccurring in the cells, the plant cells of the invention can bedifferentiated from naturally occurring plant cells particularly in thatsaid additional copy(ies) is/ (are) located at places in the genomewhere it (they) (do)/ does not occur naturally. This can, for example,be determined by means of a Southern blot analysis.

The plant cells of the invention can further be differentiated fromnaturally occurring plant cells preferably by at least one of thefollowing features: If the nucleic acid molecule is heterologousregarding the plant cell, the transgenic plant cells exhibit transcriptsof the nucleic acid molecule introduced, which can be detected by e.g.Northern blot analysis. Preferably, the plant cells of the inventioncontain a protein which is encoded by a nucleic acid moleculeintroduced. This can be detected by e.g. immunological methods,particularly by Western blot analysis.

If the nucleic acid molecule is homologue regarding the plant cell, thecells of the invention can be differentiated from naturally occurringcells, for example, due to the additional expression of the foreignnucleic acid molecules introduced. Preferably, the transgenic plantcells contain more transcripts of the foreign nucleic acid molecules.This can be detected by e.g. Northern blot analysis.

The term “genetically modified” means that the plant cell is modified inits genetic information by the introduction of a foreign nucleic acidmolecule and that the presence or the expression of the foreign nucleicacid molecule leads to a phenotypic change. In this context, phenotypicchange preferably means a measurable change of one or more functions ofthe cells. For example, the genetically modified plant cells of theinvention exhibit an increase in the activity of a plastidial ADP/ATPtranslocator due to the presence or upon expression of the foreignnucleic acid molecule introduced.

In the context of the present invention the term “increase in theactivity” means an increase of the expression of a plastidial ADP/ATPtranslocator gene, an increase in the amount of plastidial ADP/ATPtranslocator protein and/or an increase in the activity of a plastidialADP/ATP translocator in the cells.

The increase in the expression can, for example, be determined bymeasurement of the amount of transcripts encoding ADP/ATP translocator,for example by means of Northern blot analysis. In this context, anincrease preferably means an increase in the amount of transcripts incomparison with corresponding non-genetically modified cells by at least10%, preferably by at least 20%, particularly by at least 50% andparticularly preferred by at least 75%. The increase in the amount ofADP/ATP translocator protein can, for example, be determined by Westernblot analysis. In this context, an increase preferably means an increasein the amount of ADP/ATP translocator protein in comparison withcorresponding non-genetically modified cells by at least 10%, preferablyby at least 20%, particularly by at least 50% and particularly preferredby at least 75%.

The activity of the plastidial ADP/ATP translocator can be determined,for example, by isolating the plastids from the corresponding tissue anddetermining the V_(max)-values of the ATP import by means of thesilicone oil filtration method. The purification of various plastidtypes is described in e.g. Neuhaus et al. (Biochem. J. 296 (1993),395-401). The silicone oil filtration method is described e.g. in Quicket al. (Plant Physiol. 109 (1995), 113-121).

It was surprisingly found that with plants containing said plant cellswith increased activity of the plastidial ADP/ATP translocator the yieldof content substances and/or biomass is increased in comparison withcorresponding non-modified wild type plants. It was, for example, foundthat the oil content and/or the starch content in plants according tothe invention is increased and/or that also the amylose content of thesestarches is increased in comparison with non-modified wild type plants.

In this context, the term “wild type plant” refers to plants which serveas starting material for the production of the plants described, i.e.plants of which the genetic information—apart from the geneticmodification introduced—is identical to the genetic information of aplant of the invention.

The term “increased yield” means that the portion of content substances,preferably starch or oil in the plant cells of the invention isincreased by at least 10%, preferably by at least 20%, more preferablyby at least 30% and most preferably by at least 40% in comparison withplant cells of non-modified wild type plants.

The term “increased starch content” means that the portion of starch inplant cells according to the invention is increased by at least 10%,preferably by at least 20%, more preferably by at least 30% and mostpreferably by at least 40% in comparison with plant cells ofnon-modified wild type plants.

The determination of the starch portion is carried out according to themethods described in the appended Examples.

The term “increased amylose content” means that the amylose content ofthe starch synthesized in the plant cells of the invention is increasedby at least 10%, preferably by at least 20%, more preferably by at least30% and most preferably by at least 40% in comparison with plant cellsof non-modified wild type plants.

The amylose content is determined by carrying out the methods describedin the appended Examples.

As mentioned above, the plastidial ADP/ATP translocator is a transportprotein which is localized in the inner membrane of plastids (Heidt etal, FEBS Lett. 5 (1969), 11-14; Pozueta-Romero et al., Proc. Nat. Acad.Sci. USA 88 (1991), 5769-5773; Neuhaus, Plant Physiol. 101 (1993)573-578; Schünemann et al., Plant Physiol. 103 (1993), 131-137) andwhich catalyzes the transport of ATP into the plastids and of ADP out ofthe plastids Thus, the plastidial ADP/ATP translocator provides thestroma with cytosolic ATP. Kampfenkel et al. (FEBS Lett. 374 (1995),351-355) were the first to isolate a cDNA encoding an ADP/ATPtranslocator (AATP1) from Arabidopsis thaliana (Neuhaus et al., Plant J.11 (1997), 73-82) and which exhibits a great similarity (66.2%similarity) to the ADP/ATP translocator of the Gram-negative bacteriumRickettsia prowazekii. The AATP1-cDNA from A. thaliana encodes astrongly hydrophobic protein consisting of 589 amino acids whichexhibits 12 potential transmembrane helices (Kampfenkel et al., FEBSLett. 374 (1995), 351-355). Said cDNA could be functionally expressed inbakers' yeast and E. coli. After extraction of the protein andreconstitution in proteoliposomes an increase in the ATP transport ratecould be determined (Neuhaus et al., Plant J. 11 (1997), 73-82). Bymeans of antibodies against a peptide fragment of the AATP1 from A.thaliana it could be shown that the ADP/ATP translocator AATP1 islocalized in the inner chloroplast envelope membrane (Neuhaus et al.,Plant J. 11 (1997), 73-82).

The function of the plastidial ADP/ATP translocator for the plantmetabolism could not be definitely clarified so far. Various functionshave been taken into consideration, e.g. that the supply of the stromawith cytosolic ATP could have an influence on the import of proteinsinto the plastids, on the amino acid biosynthesis, the fatty acidmetabolism or the starch metabolism (Flügge and Hinz, Eur. J. Biochem.160 (1986), 563-570; Tetlow et al., Planta 194 (1994), 454-460; Hill andSmith, Planta 185 (1991), 91-96; Kleppinger-Sparace et al., PlantPhysiol. 98 (1992), 723-727).

The fact that an increase in the activity of the plastidial ADP/ATPtranslocator leads to an increase in the starch content in thecorresponding transgenic plants was, however, completely surprising.Just as surprising was the finding that the increase in the activity ofthe plastidial ADP/ATP translocator has an effect on the molecularcomposition of the produced starch. The starch from tubers of potatoplants according to the invention, for example, exhibits an increasedamylose content in comparison with starches from tubers ofnon-transformed potato plants.

So far, it has been assumed that the molecular properties of starch areexclusively determined by the interaction of starch-synthesizingenzymes, such as the branching enzymes (E.C. 2.4.1.18), the starchsynthases (E.C. 2.4.1.21) and the ADP-glucosepyrophosphorylase (E.C.2.7.7.27). The fact that the expression of a plastidial transportprotein has an influence on the structure of the starch is, however,completely surprising.

The plant cells of the invention can be derived from any plant species,i.e. both from monocotyledonous and from dicotyledonous plants.Preferably the plant cells are from agricultural crop plants, i.e. fromplants cultivated by humans for the purpose of nutrition or fortechnical, particularly industrial purposes. Generally preferred areplant cells from oil- and/or starch-synthesizing or oil- and/orstarch-storing plants. Thus, the invention preferably relates to plantcells from starch-synthesizing or starch-storing plants such as cereals(rye, barley, oat, wheat, millet, sago etc.), rice, peas, maize,medullar pea, cassava, potato, rape, soy bean, hemp, flax, sunflower orvegetables (tomato, chicory, cucumber, salad etc.). Plant cells frompotato, sunflower, soy bean, rice are preferred. Particularly preferredare plant cells from maize, wheat, rape and rice.

Furthermore, subject-matter of the invention are transgenic plantscontaining the transgenic plant cells described above. Said plants canbe produced, for example, by regeneration from plant cells of theinvention. The transgenic plants can, in principle, be plants of anyspecies, i.e. both monocotyledonous and dicotyledonous plants.Preferably, they are useful plants i.e. plants cultivated by humans forthe purpose of nutrition or for technical, particularly industrialpurposes. These plants can be oil-and/or starch-synthesizing or oil-and/or starch-storing plants. The invention preferably relates to plantssuch as cereals (rye, barley, oat, wheat, millet, sago etc.), rice,peas, maize, medullar pea, cassava, potato, rape, soy bean, hemp, flax,sunflower or vegetables (tomato, chicory, cucumber, salad etc.).Preferred are potato, sunflower, soy bean, rice. Particularly preferredare maize, wheat, rape and rice.

As mentioned before, it was surprisingly found that in starch-storingplants containing plant cells of the invention with increased activityof the plastidial ADP/ATP translocator the starch content is increasedin comparison with wild type plants and/or that also the amylose contentof these starches is increased in comparison with correspondingnon-modified wild type plants.

Thus, in a preferred embodiment the present invention also relates tostarch-storing plants which contain the plant cells of the invention andwhich exhibit an increased starch content in comparison withnon-modified wild type plants and/or an increased amylose content ofsaid starch in comparison with corresponding non-modified wild typeplants.

The term “starch-storing plants” comprises all plants withstarch-storing tissues such as maize, wheat, rice, potato, rye, barley,oat. Rice, barley and potato are preferred. Particularly preferred aremaize and wheat.

In this context, an increase in the “yield” (“increased yield”), anincrease in the starch content (“increased starch content”), an increasein the amylose content (“increased amylose content”) and the term “wildtype plant” are used within the meaning of the definitions above and areused within the same meaning for the following embodiments of theinvention, too. The term “increased yield” preferably means an increasein the production of content substances and/or biomass, in particular,if it is measured by means of fresh weight per plant.

Said increase in the yield preferably relates to parts of plants whichcan be harvested such as seeds, fruits, storage roots, roots, tubers,blossoms, buds, shoots, stems or wood.

According to the invention the increase in the yield is at least 3% withregard to the biomass and/or content substances in comparison withcorresponding non-transformed plants of the same genotype, if saidplants are cultivated under the same conditions, preferably at least10%, more preferably at least 20% and most preferably at least 30% oreven 40% in comparison with wild type plants.

Said plants according to the invention have, for example, in comparisonwith other plants synthesizing starch with increased amylose contentsuch as the amylose-extender and the dull mutants from maize, theadvantage that apart from an increased amylose content they exhibit noreduced but even an increased starch content.

Moreover, subject-matter of the present invention are oil-storing plantswhich contain the plant cells of the invention and which exhibit anincreased oil content in comparison with non-modified wild type plantcells, preferably in cells of oil-storing tissue.

The term “oil-storing plants” comprises all plants able to store oilsuch as rape, canola, soy bean, sunflower, maize, peanut, wheat, cotton,oil palms, olive trees and avocado. Preferred are maize, wheat and soybean. Particularly preferred are rape and canola.

The term “increased oil content” means that the oil content in plantcells of the invention is increased by at least 10%, preferably by atleast 20%, more preferably by at least 30% and most preferably by atleast 40% in comparison with plant cells from non-modified wild typeplants.

Methods for the determination of the oil content are known to the personskilled in the art and described, for example, by Matthaeus and Bruehl,GIT Labor-Fachz. 43 (1999), 151-152, 154-155; Matthaeus, Laborpraxis 22(1998), 52-55. The determination of the oil content may also be carriedout by non-invasive near IR-spectroscopy which is an analysing method(commonly used in breeding) and was described e.g. by Schulz et al., J.Near Infrared Spectrosc. 6 (1998), A125-A130; Starret al., J. Agric.Sci. 104 (1985), 317-323.

Plants exhibiting an increased concentration of oil are of greatcommercial interest. Maize plants, for example, whose grains exhibit ahigh level of starch but also an increased content of the side productoil are of great interest for the wet milling industry since the sideproduct is of high value. The feed-stuff industry is also interested infeeding plants with increased oil content since such plants have anincreased nutritious value. For the oil plants-processing industry anincrease of the oil content means an increase of the efficiency of theoil extracting process.

The present invention further relates to a method for the production oftransgenic plants which, compared to wild type plants, exhibit anincreased yield, wherein

-   (a) a plant cell is genetically modified by means of introduction of    a foreign nucleic acid molecule and the genetic modification leads    to an increase of the activity of a plastidial ADP/ATP translocator;    and-   (b) a plant is regenerated from the cell; and optionally-   (c) further plants are produced from the plant according to (b).

The present invention further relates to a method for the production oftransgenic plants which, compared to wild type plants, exhibit anincreased starch content and/or whose starch exhibits an increasedamylose content in comparison with corresponding wild type plants,wherein

-   (a) a plant cell is genetically modified by means of introduction of    a foreign nucleic acid molecule and the genetic modification leads    to an increase of the activity of a plastidial ADP/ATP translocator;    and-   (b) a plant is regenerated from the cell; and optionally-   (c) further plants are produced from the plant according to (b).

Moreover, subject-matter of the present invention is a method for theproduction of transgenic plants which, compared to wild type plants,exhibit an increased oil content, wherein

-   (a) a plant cell is genetically modified by means of introduction of    a foreign nucleic acid molecule and the genetic modification leads    to an increase of the activity of a plastidial ADP/ATP translocator;    and-   (b) a plant is regenerated from the cell; and optionally-   (c) further plants are produced from the plant according to (b).

For the modification introduced into the plant cell according to step(a) the same applies as has been discussed above regarding the plantcells and plants of the invention.

The regeneration of plants according to step (b) can be carried outaccording to methods known to the person skilled in the art.

The generation of further plants according to step (c) of the methods ofthe invention can be achieved e.g. by vegetative propagation (forexample via cuttings, tubers or via callus culture and regeneration ofwhole plants) or by sexual reproduction. Preferably, sexual reproductiontakes place in a controlled manner, i.e. selected plants with specificproperties are crossed with each other and propagated.

The present invention also relates to the plants obtainable by themethod of the invention.

The present invention also relates to propagation material of plantsaccording to the invention as well as of the transgenic plants producedaccording to the methods of the invention which contains geneticallymodified cells of the invention. In this context, the term propagationmaterial comprises those components of the plant which are suitable forthe generation of descendants by means of a vegetative or sexual way.Suitable for vegetative propagation are, for example, cuttings, calluscultures, rhizomes or tubers. Other propagation material comprises, forexample, fruit, seeds, seedlings, protoplasts, cell cultures, etc.Preferably, propagation material are tubers, particularly preferredseeds.

The present invention further relates to the use of nucleic acidmolecules encoding a plastidial ADP/ATP translocator for the productionof transgenic plants with an increased yield in comparison with wildtype plants.

The present invention further relates to the use of nucleic acidmolecules encoding a plastidial ADP/ATP translocator for the productionof plants which, in comparison with wild type plants, have an increasedstarch content in the starch-synthesizing and/or—storing tissue, or forthe production of plants synthesizing a starch which, compared to starchfrom wild type plants, exhibits an increased amylose content. Thenucleic acid molecules mentioned above in connection with the cells ofthe invention are preferably used.

The present invention further relates to the use of nucleic acidmolecules encoding a plastidial ADP/ATP translocator for the productionof transgenic plants which, in comparison with wild type plants, have anincreased oil content.

The present invention further relates to transgenic plant cells whichare genetically modified, wherein the genetic modification leads to thedecrease of the activity of a plastidial ADP/ATP translocator presentendogenously in the plant cell, compared to non-genetically modifiedplant cells of corresponding wild type plants.

The term “transgenic”, as used herein, means that the plant cells of theinvention deviate in their genetic information from correspondingnon-modified plant cells due to a genetic modification, particularly theintroduction of a foreign nucleic acid molecule.

In this context, the term “genetically modified” means that the plantcell is modified in its genetic information due to the introduction of aforeign nucleic acid molecule and that the presence or the expression ofthe foreign nucleic acid molecule leads to a phenotypic change.Phenotypic change preferably means a measurable change of one or morefunctions of the cell. For example, genetically modified plant cells ofthe invention exhibit a decrease of the activity of a plastidial ADP/ATPtranslocator.

The production of said plant cells of the invention with a decreasedactivity of an ADP/ATP translocator can be achieved by various methodsknown to the person skilled in the art, e.g. by methods leading to aninhibition of the expression of endogenous genes encoding a plastidialADP/ATP translocator. Such methods include, for example, the expressionof a corresponding antisense-RNA, the expression of a sense-RNA forachieving a cosuppression effect, the expression of a correspondinglyconstructed ribozyme which specifically cleaves transcripts encoding anADP/ATP translocator or the so-called “in-vivo mutagenesis”.

For the reduction of the activity of an ADP/ATP translocator in thecells of the invention preferably an antisense-RNA is expressed.

For the expression either a DNA molecule can be used comprising thewhole sequence encoding a ADP/ATP translocator including flankingsequences that are possibly present or DNA molecules comprising onlyparts of the coding sequence, wherein these parts have to be long enoughto lead to an antisense-effect in the cells. In general, sequences canbe used up to a minimum length of 15 bp, preferably a length of 100-500bp, for an efficient antisense-inhibition, particularly, sequences witha length of more than 500 bp. DNA molecules shorter than 5000 bp arecommonly used, preferably sequences shorter than 2500 bp.

It is also possible to use DNA sequences which have a high degree ofhomology to the sequences which occur endogenously in the plant cell andwhich encode a plastidial ADP/ATP translocator. The minimal homologyshould be higher than approximately 65%. The use of sequences withhomologies between 95 and 100% is to be preferred.

Alternatively, the reduction of the ADP/ATP translocator activity in theplant cells of the invention can also be accomplished by means of aco-suppression effect. The method is known to the person skilled in theart and is described, for example, in Jorgensen (Trends Biotechnol. 8(1990), 340-344), Niebel et al. (Curr. Top. Microbiol. Immunol. 197(1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197(1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995),149-159), Vaucheret et al. (Mol. Gen. Genet. 248 (1995), 311-317), deBorne et al. (Mol. Gen. Genet. 243 (1994), 613-621) and other sources.

The expression of ribozymes for the reduction of the activity ofspecific proteins in cells is also known to the person skilled in theart and is described, for example, in EP-B1 0 321 201. The expression ofribozymes in plant cells was described, for example, in Feyter et al.(Mol. Gen. Genet. 250 (1996), 329-338).

Moreover, the reduction of the ADP/ATP translocator activity in theplant cells of the invention can also be achieved by means of theso-called “in vivo mutagenesis”, wherein a hybridRNA-DNA-oligonucleotide (“chimeroplast”) is introduced into cells bymeans of transformation of cells (Kipp, P. B. et al., Poster Session atthe “5^(th) International Congress of Plant Molecular Biology, 21-27.September 1997, Singapore; R. A. Dixon and C. J. Arntzen, Meeting reporton “Metabolic Engineering in Transgenic Plants”, Keystone Symposia,Copper Mountain, Colo., USA, TIBTECH 15 (1997), 441-447; internationalpatent application WO. 95/15972; Kren et al., Hepatology 25 (1997),1462-1468; Cole-Strauss et al., Science 273 (1996), 1386-1389).

A part of the DNA component of the RNA-DNA-oligonucleotide is homologousto a nucleic acid sequence of an endogenous ADP/ATP translocator but,compared to the nucleic acid sequence of the endogenous ADP/ATPtranslocator, exhibits a mutation or contains a heterologous regionwhich is enclosed by the homologous regions.

By means of base pairing of the homologous regions of theRNA-DNA-oligonucleotide and the endogenous nucleic acid moleculefollowed by homologous recombination, the mutation or heterologousregion contained in the DNA component of the RNA-DNA-oligonucleotide canbe transferred into the genome of a plant cell. This leads to a decreaseof the activity of the plastidial ADP/ATP translocator.

Thus, subject-matter of the present invention particularly aretransgenic plant cells,

-   (a) containing a DNA molecule which can lead to the synthesis of an    antisense RNA causing a decrease of the expression of endogenous    genes which encode a plastidial ADP/ATP translocator; and/or-   (b) containing a DNA molecule which can lead to the synthesis of a    co-suppression-RNA causing a decrease of the expression of    endogenous genes which encode a plastidial ADP/ATP translocator;    and/or-   (c) containing a DNA molecule which can lead to the synthesis of a    ribozyme which can specifically cleave transcripts of endogenous    genes encoding an ADP/ATP translocator; and/or-   (d) which, due to an in vivo mutagenesis, exhibit a mutation or an    insertion of a heterologous DNA sequence in at least one endogenous    gene encoding a plastidial ADP/ATP translocator, wherein the    mutation or insertion causes a decrease of the expression of the    gene or the synthesis of an inactive transporter molecule.

The term “decrease of the activity” in the present invention means adecrease of the expression of endogenous genes encoding an ADP/ATPtranslocator, a reduction of the amount of ADP/ATP translocator proteinin the cells and/or a decrease of the biological activity of the ADP/ATPtranslocator protein in the cells.

The decrease of the expression can be determined, for example, bymeasuring the amount of transcripts encoding the ADP/ATP translocator,e.g. by Northern blot analysis. A decrease preferably means a decreaseof the amount of transcripts in comparison with genetically non-modifiedcells by at least 30%, preferably by at least 50%, more preferably by atleast 70%, particularly preferred by at least 85% and most preferably byat least 95%.

The decrease of the amount of ADP/ATP translocator protein can bedetermined, for example, by means of Western blot analysis. A decreasepreferably means a decrease of the amount of ADP/ATP translocatorprotein in comparison with corresponding genetically non-modified cellsby at least 30%, preferably by at least 50%, more preferably by at least70%, particularly preferred by at least 85% and most preferably by atleast 95%.

Surprisingly, it was found that the starch content of plant cells whichhave a decreased expression and thus a decreased activity of theplastidial ADP/ATP translocator, compared to corresponding non-modifiedplant cells from wild type plants, is reduced and that also the amylosecontent of these starches, compared to corresponding non-modified plantcells from wild type plants, is reduced. The fact that the starches ofthe plants of the invention have a modified structure is particularlysurprising since it has been assumed so far that the molecularproperties of starches are exclusively determined by the interaction ofstarch-synthesizing enzymes such as the branching enzymes (E.C.2.4.1.18) and the starch synthases (E.C. 2.4.1.21). It is completelysurprising that the expression of a plastidial transport proteininfluences the structure of starch.

The term “decreased starch content” in the present invention means thatthe content of starch in plant cells of the invention is reduced by atleast 15%, preferably by at least 30%, more preferably by at least 40%and most preferably by at least 50% in comparison with plant cells ofnon-modified wild type plants. The starch content is determinedaccording to the methods described in the Examples.

The term “decreased amylose content” means that the content of amylosein the plant cells of the invention, in comparison with plant cells ofnon-modified wild type plants, is reduced by at least 10%, preferably byat least 20%, more preferably by at least 30% and most preferably by atleast 40%. The amylose content is determined according to the methodsdescribed in the Examples.

The term “wild type plant” has the above-defined meaning.

The plant cells of the invention can derive from any plant species, i.e.both from monocotyledonous and dicotyledonous plants. Preferably theseare plant cells from agricultural crop plants, i.e. from plantscultivated by humans for the purpose of nutrition or for technical,particularly industrial purposes. Preferably, thus, the inventionrelates to plant cells from starch-synthesizing or starch-storing plantssuch as cereals (rye, barley, oat, wheat, millet, sago etc.), rice, pea,maize, medullar pea, cassava, potato, tomato, rape, soy bean, hemp,flax, sunflower, cow pea and arrowroot. Particularly preferred are plantcells from potato.

Moreover, subject-matter of the invention are transgenic plantscontaining the transgenic plant cells described above. Said plants canbe produced by regeneration from the plant cells of the invention. Thetransgenic plants can, in principle, be plants of any plant species,i.e. both monocotyledonous and dicotyledonous plants. Preferably theyare plant cells from agricultural crop plants, i.e. from plantscultivated by humans for the purpose of nutrition or for technical,particularly industrial purposes. Preferably these arestarch-synthesizing or starch-storing plants such as cereals (rye,barley, oat, wheat, millet, sago etc.), rice, pea, maize, medullar pea,cassava, potato, tomato, rape, soy bean, hemp, flax, sunflower, cow peaand arrowroot. Particularly preferred is potato.

Said plants of the invention synthesize a starch which, compared tostarch from corresponding wild type plants, exhibits a reduced amylosecontent. The terms “reduction of the amylose content” and “wild typeplants” are defined as described above.

Furthermore, the present invention also relates to a method for theproduction of transgenic plants whose starch, compared to starch fromcorresponding wild type plants, exhibits a reduced amylose contentwherein

-   (a) a plant cell is genetically modified by means of introduction of    a foreign nucleic acid molecule and the genetic modification leads    to a decrease of the activity of a plastidial ADP/ATP translocator    present endogenously in plant cells; and-   (b) a plant is regenerated from the cell produced according to step    (a); and optionally-   (c) further plants are produced from the plant produced according to    step (b).

For the modification introduced into the plant cell according to step(a) the same applies as was discussed earlier in connection with theplant cells and plants of the invention.

The regeneration of plants according to step (c) can be carried outaccording to methods known to the person skilled in the art.

The production of further plants according to step (c) of the method ofthe invention can, for example, be achieved by vegetative propagation(for example via cuttings, tubers or via callus culture and regenerationof whole plants) or by sexual propagation. Preferably, sexualpropagation takes place in a controlled manner, i.e. selected plantswith specific properties are crossed with each other and propagated.

In a preferred embodiment the method of the invention is used for theproduction of transgenic potato plants.

The present invention also relates to plants obtainable by the method ofthe invention.

The present invention also relates to propagation material of plantsaccording to the invention as well as of the transgenic plants producedaccording to the methods of the invention which contains geneticallymodified cells of the invention. In this context, the term propagationmaterial comprises those components of the plant which are suitable forthe generation of descendants by means of a vegetative or sexual way.Suitable for vegetative propagation are, for example, cuttings, calluscultures, rhizomes or tubers. Other propagation material comprises, forexample, fruit, seeds, seedlings, protoplasts, cell cultures, etc.Preferably, propagation material are seeds, particularly preferredtubers.

Furthermore, the present invention relates to the use of nucleic acidmolecules encoding a plastidial ADP/ATP translocator, of complementsthereof or of parts of said molecules for the production of plantssynthesizing a starch with, in comparison with starch from wild typeplants, reduced amylose content. Preferably, the nucleic acid moleculesmentioned above in connection with the plant cells of the inventionexhibiting an increased ADP/ATP translocator activity are to be used.

A variety of techniques are at disposal for the introduction of DNA in aplant host cell. These techniques comprise the transformation of plantcells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as transformation agent, the fusion of protoplasts, theinjection, the electroporation of DNA, the introduction of the DNA viathe biolistic approach and other possibilities.

The use of Agrobacteria-mediated transformation of plant cells has beenanalysed in detail and was described sufficiently in EP 120516; Hoekema,in: The Binary Plant Vector System Offsetdrukkerij Kanters B. V.,Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4,1-46 and An et al., EMBO J. 4 (1985), 277-287. For the transformation ofpotato, see e.g. Rocha-Sosa et al., EMBO J. 8 (1989), 29-33.

The transformation of monocotyledonous plants by means ofAgrobacterium-based vectors was also described (Chan et al., Plant Mol.Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Denget al., Science in China 33 (1990), 28-34; Wilmink et al., Plant CellReports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492;Connor and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie etal., Transgenic Res. 2 (1993), 252-265). An alternative system for thetransformation of monocotyledonous plants is the transformation via thebiolistic approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48;Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., PlantMol. Biol. 24 (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79(1990), 625-631), the protoplast transformation, the electroporation ofpartially permeabilized cells, the introduction of DNA by means of glassfibres. The transformation of maize, in particular, is described in theliterature several times (see e.g. WO 95/06128, EP 0513849, EO 0465875,EP 292435; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm etal., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11(1993), 194-200; Moroc et al., Theor. Appl. Genet. 80 (1990), 721-726).

The successful transformation of other cereals has also been described,e.g. for barley (Wan and Lemaux, loc. cit., Ritala et al., loc. cit.,Krens et al., Nature 296 (1982), 72-74) and for wheat (Nehra et al.,Plant J. 5 (1994), 285-297).

For the expression of the nucleic acid molecules encoding an ADP/ATPtranslocator in sense- or antisense-orientation in plant cells saidnucleic acid molecules are preferably linked with regulatory DNAelements which ensure the transcription in plant cells. Said elementsinclude, in particular, promoters. Generally, any promoter active inplant cells is suitable.

The promoter can be chosen in such a way that the expression takes placeconstitutively or only in a specific tissue, at a specific point in timeof the plant development or at a point in time determined by externalfactors. Both with regard to the plant and with regard to the nucleicacid molecule, the promoter can be homologous or heterologous.

Suitable promoters are e.g. the promoter of the 35S RNA of thecauliflower mosaic virus and the ubiquitin promoter from maize for aconstitutive expression, the patatin gene promoter B33 (Rocha-Sosa etal., EMBO J. 8 (1989), 23-29) for a tuber-specific expression in potatoand a promoter ensuring an expression only in photosynthetically activetissue, e.g. the ST-LS1-promoter (Stockhaus et al., Proc. Natl. Acad.Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451) or, for an endosperm-specific expression, the HMG promoterfrom wheat, the USP promoter, the phaseolin promoter, promoters fromzein genes from maize (Pedersen et al., Cell 29 (1982), 1015-1026;Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93), glutelin promoter(Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J.4 (1993), 357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218) orshrunken-1 promoter (Werr et al., EMBO J. 4 (1985), 1373-1380).Promoters which are activated only at a point in time determined byexternal factors can, however, also be used (see for example WO9307279). In this context, promoters of heat-shock proteins allowing fora simple induction can particularly be of interest. Moreover,seed-specific promoters can be used such as the USP promoter from Viciafaba which ensures a seed-specific expression in Vicia faba and otherplants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein etal., Mol. Gen. Genet. 225 (1991), 459-467).

The aforementioned embodiments with the endospecific¹ promoters aresuitable, in particular, for increasing the starch content in theendosperm. In contrast thereto, the use of embryo-specific promoters isof interest, in particular, for increasing the oil content since, as arule, oil is mainly stored in the embryo.

¹Translator's note: should read “endosperm-specific”

Thus, preferably a promoter is used according to the present inventionwhich ensures the expression in the embryo or in the seed. In apreferred embodiment of the invention the promoter is the globulin-1(glb1) promoter from maize (Styer and Cantliffe, Plant Physiol. 76(1984), 196-200). In another embodiment of the invention theembryo-specific promoter is from plants, preferably from Cuphealanceolata, Brassica repa or Brassica napus. Particularly preferred arethe promoters pCIFatB3 and pCIFatB4 (WO 95/07357). These are promotersof the genes CIFatB3 and CIFatB4, respectively, which have alreadysuccessfully been used in transgenic rape for the biosynthesis ofmedium-chain fatty acids and, thus, have a suitable expression windowfor the solution of the present problem.

In another preferred embodiment the pCIGPDH promoter (WO 95/06733), thenapin (described, for example, by Kridl, Seed Sci. Res. 1 (1991),209-219; Ellerstrom et al., Plant Mol. Biol. 32 (1996), 1019-1027;Stalberg et al., Planta 199 (1996), 515-519) or the oleosin promoter(described, for example, by Keddie, Plant Mol. Biol 24 (1994), 327-340;Plant et al., Plant Mol. Biol. 25 (1994), 193-205) is used.

Moreover, a termination sequence can be present which serves the correcttermination of the transcription and the addition of a poly-A-tail tothe transcript regarded as having a function in stabilizing thetranscripts. Said elements are described in the literature (see, e.g.,Gielen et al., EMBO J. 8 (1989), 23-29) and are interchangeable asdesired.

The transgenic plant cells and plants of the invention synthesize,preferably due to the increase or decrease of the activity of aplastidial ADP/ATP translocator, a starch which, compared to synthesizedstarch in wild type plants, is modified in its physic chemicalproperties, in particular the amylose/amylopectin ratio. In particular,said starch can, compared to wild type starch, be modified with regardto the viscosity and/or the gel formation properties of glues of saidstarch.

Thus, the present invention relates to methods for the production of amodified starch comprising the step of extraction of the starch from oneof the above-described plants and/or from starch-storing parts of saidplant. Preferably, said method comprises also the step of harvesting thecultivated plants and/or starch-storing parts of said plants before theextraction of starch and further, particularly preferred, the step ofcultivating the plants of the invention before harvesting. Methods forthe extraction of the starch from plants or the starch-storing parts ofplants are known to the person skilled in the art. Moreover, methods forthe extraction of the starch from various other starch-storing plantsare described, e.g. in “Starch: Chemistry and Technology (eds.:Whistler, BeMiller and Paschall (1994), 2^(ed) edition, Academic PressInc. London Ltd; ISBN 0-12-746270-8; see e.g. chapter XII, page 412-468:maize and sorghum starch: production; by Watson; chapter XIII, page469-479: starches from tapioca, arrowroot and sago: production; byCorbishley and Miller; chapter XIV, page 491-506: starch from wheat:production, modification and uses; by Knight and Oson; and chapter XVI,page 507 to 528: starch from rice: production and uses; by Rohmer andKlem; starch from maize: Eckhoff et al., Cereal Chem. 73 (1996) 54-57,the extraction of starch from maize to industrial standard is usuallyachieved by so-called ‘wet milling’). Appliances usually used formethods for the extraction of starch from plant material are separators,decanters, hydrocyclones, spray dryers and fluidized bed dryers.

Furthermore, subject-matter of the present invention is starchobtainable from the transgenic plant cells, plants and propagationmaterial of the invention and starch obtainable by the method of theinvention described above.

The starches of the invention can be modified according to methods knownto the person skilled in the art and are suitable for various uses inthe foodstuff or non-foodstuff industry in unmodified or modified form.

In principle, possibilities of use can be divided into two large areas.One area comprises hydrolysis products of the starch, mainly glucose andglucan building blocks obtained via enzymatic or chemical methods. Theyserve as starting material for further chemical modifications andprocesses such as fermentation. For a reduction of costs the simplicityand inexpensive carrying out of a hydrolysis method can be ofimportance. At present, the method is essentially enzymatic with use ofamyloglucosidase. It would be possible to save costs by reducing use ofenzymes. This could be achieved by changing the structure of the starch,e.g. surface enlargement of the grain, easier digestibility due to lowbranching degree or a steric structure limiting the accessibility forthe enzymes used.

The other area where starch is used as so-called native starch due toits polymeric structure can be subdivided into two further fields ofapplication:

1. Use in Foodstuffs

Starch is a classic additive for various foodstuffs, in which itessentially serves the purpose of binding aqueous additives and/orcauses an increased viscosity or an increased gel formation. Importantcharacteristic properties are flowing and sorption behaviour, swellingand pastification temperature, viscosity and thickening performance,solubility of the starch, transparency and paste structure, heat, shearand acid resistance, tendency to retrogradation, capability of filmformation, resistance to freezing/thawing, digestibility as well as thecapability of complex formation with e.g. inorganic or organic ions.

2. Use in Non-Foodstuffs

The other major field of application is the use of starch as an adjuvantin various production processes or as an additive in technical products.The major fields of application for the use of starch as an adjuvantare, first of all, the paper and cardboard industry. In this field, thestarch is mainly used for retention (holding back solids), for sizingfiller and fine particles, as solidifying substance and for dehydration.In addition, the advantageous properties of starch with regard tostiffness, hardness, sound, grip, gloss, smoothness, tear strength aswell as the surfaces are utilized.

2.1 Paper and Cardboard Industry

Within the paper production process, a differentiation can be madebetween four fields of application, namely surface, coating, mass andspraying.

The requirements on starch with regard to surface treatment areessentially a high degree of brightness, corresponding viscosity, highviscosity stability, good film formation as well as low formation ofdust. When used in coating the solid content, a corresponding viscosity,a high capability to bind as well as a high pigment affinity play animportant role. As an additive to the mass rapid, uniform, loss-freedispersion, high mechanical stability and complete retention in thepaper pulp are of importance. When using the starch in spraying,corresponding content of solids, high viscosity as well as highcapability to bind are also significant.

2.2 Adhesive Industry

A major field of application is, for instance, in the adhesive industry,where the fields of application are subdivided into four areas: the useas pure starch glue, the use in starch glues prepared with specialchemicals, the use of starch as an additive to synthetic resins andpolymer dispersions as well as the use of starches as extenders forsynthetic adhesives. 90% of all starch-based adhesives are used in theproduction of corrugated board, paper sacks and bags, compositematerials for paper and aluminum, boxes and wetting glue for envelopes,stamps, etc.

2.3 Textiles and Textile Care Products

Another possible use as adjuvant and additive is in the production oftextiles and textile care products. Within the textile industry, adifferentiation can be made between the following four fields ofapplication: the use of starch as a sizing agent, i.e. as an adjuvantfor smoothing and strengthening the burring behaviour for the protectionagainst tensile forces active in weaving as well as for the increase ofwear resistance during weaving, as an agent for textile improvementmainly after quality-deteriorating pretreatments, such as bleaching,dying, etc., as thickener in the production of dye pastes for theprevention of dye diffusion and as an additive for warping agents forsewing yarns.

2.4 Building Industry

Furthermore, starch may be used as an additive in building materials.One example is the production of gypsum plaster boards, in which thestarch mixed in the thin plaster pastifies with the water, diffuses atthe surface of the gypsum board and thus binds the cardboard to theboard. Other fields of application are admixing it to plaster andmineral fibers. In ready-mixed concrete, starch may be used for thedeceleration of the sizing process.

2.5 Ground Stabilisation

Furthermore, the starch is advantageous for the production of means forground stabilisation used for the temporary protection of groundparticles against water in artificial earth shifting. According tostate-of-the-art knowledge, combination products consisting of starchand polymer emulsions can be considered to have the same erosion- andencrustation-reducing effect as the products used so far; however, theyare considerably less expensive.

2.6 Use in Plant Protectives and Fertilizers

Another field of application is the use of starch in plant protectivesfor the modification of the specific properties of these preparations.For instance, starch is used for improving the wetting of plantprotectives and fertilizers, for the dosed release of the activeingredients, for the conversion of liquid, volatile and/or odorousactive ingredients into microcristalline, stable, deformable substances,for mixing incompatible compositions and for the prolongation of theduration of the effect due to a reduced disintegration.

2.7 Drugs, Medicine and Cosmetics Industry

Starch may also be used in the fields of drugs, medicine and in thecosmetics industry. In the pharmaceutical industry, starch may be usedas a binder for tablets or for the dilution of the binder in capsules.Furthermore, starch is suitable as disintegrant for tablets since, uponswallowing, it absorbs fluid and after a short time it swells so muchthat the active ingredient is released. For qualitative reasons, medicallubricating and vulnerary dusting powders are further fields ofapplication. In the field of cosmetics, the starch may for example beused as a carrier of powder additives, such as scents and salicylicacid. A relatively extensive field of application for the starch istoothpaste.

2.8 Starch as an Additive in Coal and Briquettes

Starch can also be used as an additive in coal and briquettes. By addingstarch, coal can be quantitatively agglomerated and/or briquetted inhigh quality, thus preventing premature disintegration of thebriquettes. Barbecue coal contains between 4 and 6% added starch,calorated coal between 0.1 and 0.5%. Furthermore, starch is suitable asa binding agent since adding it to coal and briquette can considerablyreduce the emission of toxic substances.

2.9 Processing of Ore and Coal Slurry

Furthermore, starch may be used as a flocculant in the processing of oreand coal slurry.

2.10 Additive for Casting Materials

Another field of application is the use as an additive to processmaterials in casting. For various casting processes cores produced fromsands mixed with binding agents are needed. Nowadays, the most commonlyused binding agent is bentonite mixed with modified starches, mostlyswelling starches.

The purpose of adding starch is increased flow resistance as well asimproved binding strength. Moreover, swelling starches may fulfil moreprerequisites for the production process, such as dispersability in coldwater, rehydratisability, good mixability in sand and high capability ofbinding water.

2.11 Rubber Industry

In the rubber industry starch may be used for improving the technicaland optical quality. Reasons for this are improved surface gloss, gripand appearance. For this purpose, starch is dispersed on the stickyrubberised surfaces of rubber substances before the cold vulcanization.It may also be used for improving the printability of rubber.

2.12 Production of Leather Substitutes

Another field of application for modified starch is the production ofleather substitutes.

2.13 Starch in Synthetic Polymers

In the plastics market the following fields of application are emerging:the integration of products derived from starch into the processingprocess (starch is only a filler, there is no direct bond betweensynthetic polymer and starch) or, alternatively, the integration ofproducts derived from starch into the production of polymers (starch andpolymer form a stable bond).

The use of the starch as a pure filler cannot compete with othersubstances such as talcum. This situation is different when the specificstarch properties become effective and the property profile of the endproducts is thus clearly changed. One example is the use of starchproducts in the processing of thermoplastic materials, such aspolyethylene. Thereby, starch and the synthetic polymer are combined ina ratio of 1:1 by means of coexpression to form a ‘master batch’, fromwhich various products are produced by means of common techniques usinggranulated polyethylene. The integration of starch in polyethylene filmsmay cause an increased substance permeability in hollow bodies, improvedwater vapor permeability, improved antistatic behaviour, improvedanti-block behaviour as well as improved printability with aqueous dyes.

Another possibility is the use of the starch in polyurethane foams. Dueto the adaptation of starch derivatives as well as due to theoptimisation of processing techniques, it is possible to specificallycontrol the reaction between synthetic polymers and the hydroxy groupsof the starch. The results are polyurethane films having the followingproperty profiles due to the use of starch: a reduced coefficient ofthermal expansion, decreased shrinking behaviour, improvedpressure/tension behaviour, increased water vapour permeability withouta change in water acceptance, reduced flammability and cracking density,no drop off of combustible parts, no halides and reduced aging.Disadvantages that presently still exist are reduced pressure and impactstrength.

Product development of film is not the only option. Also solid plasticsproducts, such as pots, plates and bowls can be produced by means of astarch content of more than 50%. Furthermore, the starch/polymermixtures offer the advantage that they are much easier biodegradable.

Furthermore, due to their extreme capability to bind water, starch graftpolymers have gained utmost importance. These are products having abackbone of starch and a side lattice of a synthetic monomer grafted onaccording to the principle of radical chain mechanism. The starch graftpolymers available nowadays are characterised by an improved binding andretaining capability of up to 1000 g water per g starch at a highviscosity. These super absorbers are used mainly in the hygiene field,e.g. in products such as nappies and sheets, as well as in theagricultural sector, e.g. in seed pellets.

What is decisive for the use of the novel starch modified by recombinantDNA techniques are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ashes/phosphate content,amylose/amylopectin ratio, distribution of the relative molar mass,branching degree, granule size and shape as well as crystallization, andon the other hand, the properties resulting in the following features:flow and sorption behaviour, pastification temperature, viscosity,thickening performance, solubility, paste structure, transparency, heat,shear and acid resistance, tendency to retrogradation, capability of gelformation, resistance to freezing/thawing, capability of complexformation, iodine binding, film formation, adhesive strength, enzymestability, digestibility and reactivity.

The production of modified starch by genetically operating with atransgenic plant may modify the properties of the starch obtained fromthe plant in such a way as to render further modifications by means ofchemical or physical methods superfluous. On the other hand, thestarches modified by means of recombinant DNA techniques might besubjected to further chemical modification, which will result in furtherimprovement of the quality for certain of the above-described fields ofapplication. These chemical modifications are principally known. Theseare particularly modifications by means of

-   heat treatment-   acid treatment-   oxidation and-   esterification    leading to the formation of phosphate, nitrate, sulfate, xanthate,    acetate and citrate starches. Other organic acids may also be used    for the esterification:-   formation of starch ethers    -   starch alkyl ether, O-allyl ether, hydroxylalkyl ether,        O-carboxylmethyl ether, N-containing starch ethers, P-containing        starch ethers and S-containing starch ethers.    -   formation of branched starches    -   formation of starch graft polymers.

FIG. 1 schematically illustrates the plasmid pJT31 (AATP1 (Arabidopsisthaliana) sense);

FIG. 2 schematically illustrates the plasmid pJT32 (AATP1 (Solanumtuberosum) antisense);

FIG. 3 shows the comparison of the amino-acid sequence of the AATP2(SEQID NO:6) from Arabidopsis thaliana with the AATP1 (A. thaliana) (SEQ IDNO:5) and a homologous protein from Rickettsia prowazekii (SEQ ID NO:7)Williamson et al., Gene 80 (1989), 269-278);

FIG. 4 hydropathy analysis of AATP2 (A. thaliana), AATP1 (A. thaliana)and the Rickettsia ADP/ATP translocator carried out according to themethod of von Heijne et al. (Eur. J. Biochem. 180 (1989), 535-545)

FIG. 5 shows a Northern blot analysis of the expression of the AATP1(Solanum tuberosum) in leaf and tuber of ADP/ATP translocator antisenseplants.

FIG. 6 shows a Northern blot analysis of the expression of the AATP1(Arabidopsis thaliana) in leaf and tuber of ADP/ATP translocatoroverexpression plants.

FIG. 7 Schematic map of the cassette pTE200 for the embryo-specific geneexpression. EcoRI, SmaI, BamHI, XhoI, NotI, XbaI, SacI, KpnI, ApaI, SalIand SfiI mark recognition sites for restriction endonucleases. Forpractical reasons, SfiI (A) and SfiI (B) differ in the variablenucleotide sequence within the recognition sequence. The abbreviationsencode as follows: PCIFatB4=CIFatB4 promoter, tCIFatB4=CIFatB4terminator, amp=bacterial resistance against ampicillin, ColE1ori=“origin of replication” from the plasmid ColE1, f1(−) ori=“origin ofreplication” from the phage f1.

FIG. 8 Schematic map of the ADP/ATP translocator expression cassettepTE208: this derivative of the vector pTE200 (FIG. 7) carries a cDNAcoding for a plastidial ADP/ATP translocator from Solanum tuberosum insense-orientation.

FIG. 9 Schematic map of the binary vector pMH000-0. SfiI, SalI, ClaI,HindIII, EcoRI, NsiI, SmaI, BamHI, SpeI, NotI, KpnI, BglII, ApaI, XhoI,XbaI and BstEII mark recognition sites for restriction endonucleases.SfiI (A) and SfiI (B) differ in the variable nucleotide sequence oftheir recognition sequence as stated. This is the reason why arecircularization of the starting plasmid is prevented after SfiIcleavage and a directed insertion of the expression cassette from thepTE200 derivative is possible. The abbreviations encode as follows: RB,LB=right and left border region, t35S—termination signal of the 35S rnagene from CaMV, pat=phophinotricin acetyl transferase gene,p35S=promoter of the 35S rna gene from CaMV, p35S(min)=minimal promoterof the 35S rna gene from CaMV, tp-sul=sulfonamid resistance gene withtransit peptide, tnos=termination signal of the nopalin synthase gene,Sm/Sp=bacterial resistance against streptomycin and spectinomycin, parA,parB and parR=plasmid multiplication functions from the plasmid pVS1with large host area i.a. for Agrobacterium tumefaciens and Escherichiacoli.

The following examples illustrate the invention.

Example 1

Construction of the Bacterial Expression Vector pJT118 andTransformation of E. coli

The AATP2 protein (gene library X94626) from Arabidopsis thaliana wasN-terminally fused with a “histidine-tag” comprising 10 aminos acids.

For this, the cDNA encoding the whole AATP2 protein from Arabidopsisthaliana was isolated by means of a PCR approach. The followingoligonucleotide served as sense-primer which, in addition, had aXhoI-restriction site: cgtgagagatagagagctcgagggtctgattcaaacc (SEQ ID NO:1); comprising the base pairs 66-102).

An oligonucleotide carrying an additional BamHI-restriction site servedas antisense-primer gatacaacaggaatcctggatgaagc (SEQ ID NO: 2);comprising the base pairs 1863-1835). The obtained PCR product waspurified by means of an agarose gel, cut with the restriction enzymesXhoI/BamHI and introduced “in frame” in the plasmid pET16b (Novagene,Heidelberg, Germany). This led to the exhibition of a histidine-tag of10 amino acids at the N-terminus of the cDNA encoding the whole AATP2protein from Arabidopsis thaliana (His-AATP2). Said vector was calledpJT 118. The sequence of the PCR product was determined by sequencing ofboth nucleotide strands (Eurogentec). The transformation of E. coli C43(Miroux and Walker, J. Mol. Biol. 260 (1996), 289-298) was carried outaccording to standard methods. The E. coli strain C43 allows for theheterologous expression of animal (Miroux and Walker, loc. cit.) andplant (Tjaden et al., J. Biol. Chem. (1998) (in press)) membraneproteins.

After transformation of said strain with the vector pJT118 uptakestudies with radioactively marked ADP and ATP were carried out. It couldbe demonstrated by these studies that His-AATP2 can be functionallyexpressed in E. coli C43 in the cytoplasmic membrane of E. coli. Thisshowed that AATP2 in fact encodes an ADP/ATP translocator. The presenceof a N-terminal histidine-tag leads to an increase (2×-3×) of thetransport activity of AATP2 from A. thaliana in E. coli in comparisonwith AATP2 without N-terminal his-tag.

Example 2

Construction of the Plasmid pJT31 and Introduction of the Plasmid intothe Genome of Potato Plants

For the construction of a plant transformation vector an EcoRV/BamHIfragment of the AATP1-cDNA from A. thaliana (Kampfenkel et al., FEBSLetters 374 (1995), 351-355) with a length of 2230 bp was ligated intothe vector pBinAR cut with SmaI/EcoRV and BamHI (Hofgen and Willmitzer,Plant Sci. 66 (1990), 221-230). By insertion of the cDNA fragment anexpression cassette is formed (pJT31) which is constructed of thefragments A, B and C as follows (see FIG. 1):

Fragment A (540 bp) contains the 35S promoter from the cauliflowermosaic virus. Fragment B contains, in addition to the flanking regions,the protein-encoding region of an ADP/ATP translocator from A. thaliana(AATP1). Said region was isolated as described above and fused insense-orientation to the 35S promoter in pBinAR. Fragment C (215 bp)contains the polyadenylation signal of the octopine synthase gene fromAgrobacterium tumefaciens.

The size of the plasmid pJT31 is approximately 14.2 kb.

The plasmid was transferred into potato plants by means of Agrobacteriaas by Rocha-Sosa et al. (EMBO J. 8 (1989), 23-29). As a result of thetransformation transgenic potato plants exhibited an increase of themRNA of a plastidial ADP/ATP translocator. This was detected by Northernblot analysis (see FIG. 6). RNA was isolated according to standardprotocols from leaf and tuber tissue from potato plants. 50 μg RNA wereseparated on an agarose gel (1.5% agarose, 1×MEN puffer, 16.6%formaldehyde). After electrophoresis the RNA was transferred with 20×SSConto a nylon membrane Hybond N (Amersham, UK) by means of capillaryblot. The RNA was fixed on the membrane by means of UV irradiation. Themembrane was pre-hybridized for 2 hours in phosphate hybridizationbuffer (Sambrook et al., loc. cit.) and subsequently hybridized for 10hours by means of addition of the radioactively labeled probe.

Example 3

Construction of the Plasmid pJT32 and Introduction of the Plasmid intothe Genome of Potato Plants

For the construction of a plant transformation vector a BamHI/NdeIfragment of the coding region of the AATP1-cDNA from S. tuberosum(Genbank Y10821) with a length of 1265 bp was ligated into the vectorpBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230) cut withSmaI/NdeI and BamHI.

By insertion of the cDNA fragment an expression cassette is formed whichis constructed of the fragments A, B and C as follows (see FIG. 2):

Fragment A (540 bp) contains the 35S promoter from the cauliflowermosaic virus. Fragment B contains contains a region of an ADP/ATPtranslocator from S. tuberosum (AATP1 S.t.) with a length of 1265 bp.This region was fused in antisense-orientation to the 35S promoter inpBinAR.

Fragment C (215 bp) contains the polyadenylation signal of the octopinesynthase gene from Agrobacterium tumefaciens.

The size of the plasmid pJT32 is approximately 13.3 kb.

The plasmid was transferred into potato plants by means of Agrobacteriaas by Rocha-Sosa et al. (EMBO J. 8 (1989), 23-29).

As a result of the transformation transgenic potato plants exhibited adecrease of the mRNA of a plastidial ADP/ATP translocator. This wasdetected by Northern blot analysis (see FIG. 5). RNA was isolatedaccording to standard protocols from leaf and tuber tissue from potatoplants. 50 μg RNA were separated on an agarose gel (1.5% agarose, 1×MENpuffer, 16.6% formaldehyde). After electrophoresis the RNA wastransferred with 20×SSC onto a nylon membrane Hybond N (Amersham, UK) bymeans of capillary blot. The RNA was fixed on the membrane by means ofUV irradiation. The membrane was pre-hybridized for 2 hours in phosphatehybridization buffer (Sambrook et al., loc. cit.) and subsequentlyhybridized for 10 hours by means of addition of the radioactivelylabeled probe.

Example 4

Analysis of the Starch, Amylose and Sugar content of Transgenic PotatoPlants

The determination of the content of soluble sugars was carried asdescribed by Lowry and Passonneau in “A Flexible System of EnzymaticAnalysis”, Academic Press, New York, USA (1972). The determination ofthe starch content was carried out as described by Batz et al. (PlantPhysiol. 100 (1992), 184-190).

TABLE 1 starch in soluble sugars in (μmolC6units/g (μmol/g line/genotypefresh weight) fresh weight) Desiree/WT 1094.0 26.49 654/antisense-AATP1 574.2 42.52 (S. tuberosum) 594/antisense-AATP1  630.2 48.76 (S.tuberosum) 595/antisense-AATP1  531.4 45.92 (S. tuberosum)676/antisense-AATP1  883.0 40.60 (S. tuberosum) 62/sense-AATP1 1485.030.65 (A. thaliana) 98/sense-AATP1 1269.0 18.28 (A. thaliana)78/sense-AATP1  995.0 20.50 (A. thaliana)

The determination of the amylose content was carried out as described byHovenkamp-Hermelink et al. (Potato Res. 31 (1988), 241-246):

line/genotype % amylose Desiree/WT 18.8 654/antisense-AATP1 15.5 (S.tuberosum) 594/antisense-AATP1 14.3 (S. tuberosum) 595/antisense-AATP118.0 (S. tuberosum) 676/antisense-AATP1 11.5 (S. tuberosum)62/sense-AATP1 27.0 (A. thaliana) 98/sense-AATP1 22.7 (A. thaliana)78/sense-AATP1 24.5 (A. thaliana)

Example 5

Production of an Expression Cassette and Transformation of Rape Plants

The expression cassette pTE200 in a pBluescript derivative (Short etal., Nucl. Acid Res. 16, (1988), 7583-7600) carries the promoter andterminator sequences of the thioesterase gene CIFatB4 (GenBankaccession: AJ131741) from Cuphea lanceolata and suitable polylinkersequences for the insertion of various useful genes. Peripheral SfiIrecognition sites with non-compatible nucleotides in the variablerecognition regions allow for a directed transfer of the wholeexpression cassette including the useful gene into the correspondingrestriction sites of the binary plasmid vector pMH000-0, a furtherdevelopment of pLH9000 (Hausmann and Töpfer, (1999): 9^(th) chapter:“Entwicklung von Plasmid-Vektoren” in Bioengineering für Rapssorten nachMaβ, D. Brauer, G. Röbbelen and R. Töpfer (eds.), Vorträge fürPlanzenzüchtung, Volume 45, 155-172), and prevent recircularization ofthe DNA in the recipient vector.

For the production of the expression cassette pTE200, first, a SalI-BbvIfragment carrying a promoter with an approximate length of 3.3 kb wasisolated from the genomic clone CITEg16 (WO95/07357) carrying thecomplete CIFatB4 gene from C. lanceolata. In order to achieve this, theBbvI restriction site at the 3′-end of the promoter was opened andmodified in such a way that the fragment could then be taken up by thepBluescript (stratagene) cut with SalI and SmaI. An internal EcoRrestriction site of the fragment located 1211 nucleotides 5′ was deletedby being opened, modified by means of T4 polymerase and subsequentlyclosed again.

The terminator sequence was amplified by means of the polymerase chainreaction and specific oligonucleotide primers at the CITEg16 matrix(WO95/07357) and provided with various polylinker restriction sites(MCS) via the primers. The sequences of the primers are:5′GAATTCCTGCAGCCCGGGGGATCCACTAGTCTCGAGAAGTGGCTGGGGGCCT TTCC3′ (SEQ IDNO: 3)=5′-primer: (MCS: EcoRI, PstI, SmaI, BamHI, SpeI XhoI; CIFatB4terminator: from pos. 35-56) and5′TCTAGAGGCCAAGGCGGCCGCTTCAACGGACTGCAGTGC3′ (SEQ ID NO: 4)=3′-primerCIFatB4 terminator: from pos. 22-39, MCS: NotI, StyI, SfiI, XbaI. Theamplificate was cut with EcoRI and NotI and inserted into thecorresponding restriction sites of the pBlueSfi BA (Hausmann and Töpfer,see above). The fragment carrying the promoter was opened with BamHI,modified and subsequently cut with SalI to place it in the pBlueSfi BAvector via SalI and modified HindlII restriction site in front of theterminator. The result is the expression cassette pTE200 (see FIG. 7).For the contruction of a plant transformation vector an EcoRI fragmentof the AATP1 cDNA from Solanum tuberosum (pTM1, Tjaden et al., The PlantJournal 16 (1998) 531-540) with a length of 2270 bp was ligated into thevector pTE200 opened with EcoRI. The orientation was controlled by meansof restriction digest. The result was the plasmid pTE208 (FIG. 8). Inthe following step, the SfiI fragment from pTE208 was inserted into thepolylinker restriction sites of the binary vector pMH000-0 (FIG. 9) in adirected manner. The result was the vector pMH 0208.

The binary plasmid vector pMH000-0 has been developed further frompLH9000 (Hausmann and Töpfer, see above) with alternative selectionmarkers for the plant transformation. The sulfonamide gene (sul) wasisolated together with the signal peptide sequence (tp) for plastidialimport of the small subunit of the ribulosebiphosphate carboxylase fromthe precursor plasmid of pS001 (Reiss et al., Proc. Natl. Acad. Sci. USA93, (1996), 3094-3098) after modification of the Asp718-to theXhoI-restriction site. The XhoI-SalI fragment was inserted into theXhoI— and BamHI-restriction sites of a pBluescript derivative in frontof the terminator of the nopalin synthase gene (pAnos) aftermodification of SalI and BamHI. In a subsequent three fragment ligationthe resulting tpsul-pAnos fragment (XhoI-XbaI) and the XhoI-HindlIIfragment from pRT103pat (Töpfer et al., Methods in Enzymol. 217, (1993),66-78) were united with the plasmid pK18 (Pridmore, Gene 56, (1987),309-312) opened by means of HindlII and XbaI. As a result the gene forthe phosphinotricin acetyltransferase with the terminator of the CaMV35Srna gene from pRT103pat was placed in opposite orientation to thetpsul-pAnos unit. A dual promoter of the CaMV35S rna gene as XhoIfragment from a descendant of pROA93 (Ott et al., Mol. Gen. Genet. 221,(1990), 121-124) was inserted into the XhoI restriction site between theresistance-mediating gene sequences to complete said double selectionunit (for resistance against the herbicide Basta and the sulfonamidesulfadiazin). After corresponding modifications in the adjacentpolylinker the resulting dual selection cassette was exchanged by meansof XbaI and HindlII against the kanamycin cassette in the pLH9000precursor plasmid (Hausmann and Töpfer, see above). The result was thebinary plasmid vector pMH000-0.

The transformation of hypocotyl explants of rape of the variety Drakkarwas carried out according to the protocol of De Block (Plant Physiol. 91(1989), 694-701) by means of Agrobacteria (strain GV 3101 C58C1 Rifr)carrying the binary vector pMH0208 (ATP/ADP transporter sense). Shootswere regenerated on selective nutrient medium (sulfonamide) andcultivated in the greenhouse up to seed maturation. By means of PCR andleaf test (tolerance against glufosinatammonium (Basta®)) it was testedwhich plants contained the transgene. Maturing embryos at variousdevelopmental stages were harvested from said plants and stored inliquid nitrogen.

For the determination of the oil content mature seeds of transgenic rapelines and of control lines were analysed by means of the non-invasivenear infrared spectroscopy (described, for example, by Schulz et al., J.Near Infrared Spectrosc. 6, (1998), A125-A130; Starr et al., J. Agric.Sci. 104(2), (1985), 317-323).

1. A genetically modified plant cell wherein a foreign nucleic moleculeencoding a plastidial ADP/ATP translocator is integrated into thenuclear genome of said genetically modified plant cell and wherein theexpression of said foreign nucleic acid molecule results in an increasein plastidial ADP/ATP translocator activity in comparison withcorresponding non-genetically modified plant cells from wild typeplants.
 2. The genetically modified plant cell according to claim 1exhibiting an increased starch content in comparison with correspondingnon-genetically modified plant cells.
 3. The genetically modified plantcell according to claim 1 synthesizing a starch fraction exhibiting anincreased amylose-content in comparison with a starch fraction fromcorresponding non-genetically modified plant cells.
 4. A geneticallymodified plant containing transgenic plant cells according to claim 1.5. The genetically modified plant according to claim 4, which is amaize, wheat or potato plant.
 6. A method for the production of atransgenic plant exhibiting an increased yield in comparison with wildtype plants, wherein (a) a plant cell is genetically modified byintegrating a foreign nucleic acid molecule encoding a plastidialADP/ATP translocator into the nuclear genome of said plant cell whereinthe expression of said foreign nucleic acid molecule results in anincrease in plastidial ADP/ATP translocator activity in the cell; (b) aplant is regenerated from the cell produced according to step (a); and(c) further transgenic plants are optionally produced from the plantproduced according to step (b).
 7. The method according to claim 6,wherein the transgenic plant exhibits (a) an increased starch content incomparison with wild type plants, (b) a starch fraction with anincreased amylose content in comparison with a starch fraction from thewild type plants, or (c) both an increased starch content in comparisonwith wild type plants and a starch fraction with an increased amylosecontent in comparison with starch friction from wild type plants.
 8. Atransgenic plant obtainable by the method according to claim 6 or
 7. 9.Propagation material of genetically modified plants according to any oneof claim 4 or 5, wherein the propagation material has ADP/ATPtranslocator activity.
 10. A method for the production of a modifiedstarch comprising the extraction of the starch from a plant according toany one of claim 4 or
 5. 11. The genetically modified plant cellaccording to claim 1 exhibiting an increased starch content incomparison with corresponding non-genetically modified plant cells. 12.The method according to claim 6, wherein the transgenic plant exhibitsan increased starch content in comparison with wild type plants and astarch fraction with an increased amylose content in comparison with astarch fraction from wild type plants.
 13. The method according to claim6, wherein the transgenic plant exhibits an increased starch content incomparison with wild type plants or a starch fraction with an increasedamylose content in comparison with a starch fraction from wild typeplants.
 14. A transgenic plant produced by the method according to claim6.
 15. A genetically modified plant cell wherein a foreign nucleicmolecule encoding an Arabidopsis thaliana, Solanum tuberosum, Rickettsiaprowazekii or Chlamydia trachomatis plastidial ADP/ATP translocator isintegrated into the nuclear genome of said genetically modified plantcell and wherein the expression of said foreign nucleic acid moleculeresults in an increase in plastidial ADP/ATP translocator activity incomparison with corresponding non-genetically modified plant cells fromwild type plants.
 16. A genetically modified plant cell wherein aforeign nucleic molecule encoding a plastidial ADP/ATP translocatorprotein having an amino acid sequences of SEQ ID NO. 5, SEQ ID NO. 6 orSEQ ID NO. 7 is integrated into the nuclear genome of said geneticallymodified plant cell and wherein the expression of said foreign nucleicacid molecule results in an increase in plastidial ADP/ATP translocatoractivity in comparison with corresponding non-genetically modified plantcells from wild type plants.
 17. A method for the production of atransgenic plant exhibiting an increased starch, increased amylose, orincreased starch and amylose in comparison with wild type plants,wherein (a) a plant cell is genetically modified by integrating aforeign nucleic acid molecule encoding an Arabidopsis thaliana, Solanumtuberosum, Rickettsia prowazekii or Chlamydia trachomatis plastidialADP/ATP translocator into the nuclear genome of said plant cell whereinthe expression of said foreign nucleic acid molecule results in anincrease in plastidial ADP/ATP locator activity in the cell; (b) a plantis regenerated from the cell produced according to step (a); and (c)further transgenic plants are optionally produced from the plantproduced according to step (b).
 18. A method for the production of atransgenic plant exhibiting an increased starch, increased amylose, orincreased starch and amylose in comparison with wild type plants,wherein (a) a plant cell is genetically modified by integrating aforeign nucleic acid molecule encoding a plastidial ADP/ATP translocatorprotein having an amino acid sequence of SEQ ID NO. 5, SEQ ID NO. 6 orSEQ ID NO. 7 into the nuclear genome of said plant cell wherein theexpression of said foreign nucleic acid molecule results in an increasein plastidial ADP/ATP translocator activity in the cell; (b) a plant isregenerated from the cell produced according to step (a); and (c)further transgenic plants are optionally produced from the plantproduced according to step (b).
 19. A method for the production of atransgenic plant exhibiting an increased starch, increased amylose, orincreased starch and amylose in comparison with wild type plants,wherein (a) a plant cell is genetically modified by integrating aforeign nucleic acid molecule encoding a plastidial ADP/ATP translocatorprotein whose amino acid sequence is at least 66% homologous to SEQ IDNO. 5 into the nuclear genome of said plant cell wherein the expressionof said foreign nucleic acid molecule results in an increase inplastidial ADP/ATP translocator activity in the cell; (b) a plant isregenerated from the cell produced according to step (a); and (c)further transgenic plants are optionally produced from the plantproduced according to step (b).
 20. A genetically modified plant cellwherein a foreign nucleic molecule encoding a plastidial ADP/ATPtranslocator protein whose amino acid sequence is at least 66%homologous to SEQ ID NO. 5 is integrated into the nuclear genome of saidgenetically modified plant cell and wherein the expression of saidforeign nucleic acid molecule results in an increase in plastidialADP/ATP translocator activity in comparison with correspondingnon-genetically modified plant cells from wild type plants.